Difference between revisions of "Part:BBa K1182000:Experience"

(→‎Result)
(→‎Experimental setup & protocol)
Line 9: Line 9:
  
  
−
== '''Experimental setup & protocol''' ==
+
=='''Stability Assay of The Split Reporter'''==
  
 +
We perform a stability assay to test the enzyme’s activity after freezing and storage in 4ᵒ C freezer for a different length of time.
  
−
1. The alpha and omega fragment of β-galactosidase was cloned into pQE-80L and pQE-81L, respectively
 
  
−
2. The alpha and omega fragment was then expressed in TOP10 E.coli
+
'''Experimental setup & protocol'''
  
−
3. An equal molar of both the alpha and omega peptide, as well as the diluted full length β-galactosidase, then added into an eppendorf tube and incubated at room temperature on an orbital rocker for 1 hour.  
+
1. Add α and ω fragment of β-galactosidase to different eppendorf tube.
  
−
4. At time zero, 20µL of ONPG (4mg/mL) was added into each tube
+
2. Store those two tubes in 4ᵒC freezer for different length of time (2 days, 7 days, 14 days, 21 days and 28 days)
  
−
5. The eppendorf tubes then incubated at room temperature for different length of time (30 min, 90 min, 3 hours, and 19 hours).
+
3. Thaw the tube after stored in different length of time
  
−
6. The reaction was then terminated by adding 50µL 1M Na2CO3
+
4. Add an equal molar of both the α and ω peptide to an eppendorf tube. Incubate those tubes at room temperature on an orbital rocker for 1 hour.  
  
−
7. The absorbance is the analysed using 420 nm light
+
5. At time zero, 20µL of ONPG (4mg/mL) was added into each tube
 +
 
 +
6. The eppendorf tubes then incubated at room temperature for 3 hours
 +
 
 +
7. The reaction was then terminated by adding 50µL 1M Na2CO3
 +
 
 +
8. The absorbance is the analysed using 420 nm light  
 +
 
 +
9. Results are expressed as percent signals obtained from freshly expressed enzyme after 3 hours of reaction
  
  
  
 
'''Result'''
 
'''Result'''
 +
 +
[[File:Stability Assay.jpg]]
 +
  
  
−
              [[File:Activity Assay.jpg]]
+
'''Interpretation'''
  
−
===User Reviews===
+
The activity of beta-galactosidase enzyme is still above 95% after 28 days of storage. This data suggest that split reporter can be stored for a quite long time in 4ᵒC.
−
<!-- DON'T DELETE --><partinfo>BBa_K1182000 StartReviews</partinfo>
+
−
<!-- Template for a user review
+
−
{|width='80%' style='border:1px solid gray'
+
−
|-
+
−
|width='10%'|
+
−
<partinfo>BBa_K1182000 AddReview number</partinfo>
+
−
<I>Username</I>
+
−
|width='60%' valign='top'|
+
−
Enter the review inofrmation here.
+
−
|};
+
−
<!-- End of the user review template -->
+
−
<!-- DON'T DELETE --><partinfo>BBa_K1182000 EndReviews</partinfo>
+

Revision as of 17:53, 27 September 2013

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

===Applications of BBa_K1182000===

To test the function of our split reporter, we collect the data of enzyme activity over time. We exploit the enzyme’s ability to convert ortho-Nitrophenyl-β-galactoside (ONPG), a colorless substance, into ortho-nitrophenol, a yellow substance. We then use a spectrophotometer to quantisize the absorbance of the solution.


Stability Assay of The Split Reporter

We perform a stability assay to test the enzyme’s activity after freezing and storage in 4ᵒ C freezer for a different length of time.


Experimental setup & protocol

1. Add α and ω fragment of β-galactosidase to different eppendorf tube.

2. Store those two tubes in 4ᵒC freezer for different length of time (2 days, 7 days, 14 days, 21 days and 28 days)

3. Thaw the tube after stored in different length of time

4. Add an equal molar of both the α and ω peptide to an eppendorf tube. Incubate those tubes at room temperature on an orbital rocker for 1 hour.

5. At time zero, 20µL of ONPG (4mg/mL) was added into each tube

6. The eppendorf tubes then incubated at room temperature for 3 hours

7. The reaction was then terminated by adding 50µL 1M Na2CO3

8. The absorbance is the analysed using 420 nm light

9. Results are expressed as percent signals obtained from freshly expressed enzyme after 3 hours of reaction


Result

Stability Assay.jpg


Interpretation

The activity of beta-galactosidase enzyme is still above 95% after 28 days of storage. This data suggest that split reporter can be stored for a quite long time in 4ᵒC.